Epigenetische Veränderungen in den IL-1beta und TNFalpha Promotoren während der Zelldifferenzierung und unter Zinkmangel

IL-1beta and TNFalpha are involved in a variety of immunological processes. Their deregulation can cause immunosuppression or chronic inflammation. Both cytokines are primarily synthesized by monocytes but not neutrophil granulocytes (PMN) or their common haematopoietic precursors (HPC), as demonstrated in this thesis. Via in vitro differentiation of HPC it could be shown for the first time in primary cells that the proximal IL-1beta promoter is remodeled into an open structure during monopoiesis but not granulopoiesis. In contrast, chromatin remodeling of the TNFalpha promoter was stimulation dependent, but restricted to monocytes, only. No changes in TNFalpha promoter structure were detected in stimulated PMN or HPC. Basis for the chromatin remodeling of IL-1beta and TNFalpha promoters is presumably their hyperacetylation, simulated by inhibition of histone deacetylases (HDAC) herein. Specific DNA- or histone methylation of IL-1beta and TNFalpha promoter during differential activation of IL-1beta and TNFalpha expression of monocytes and PMN could be excluded. However, an indirect effect due to methylation dependent activation of IL-1beta and TNFalpha regulating genes is assumable. The involvement of the myeloid transcription factors PU.1 and C/EBPbeta is also unlikely, since their binding pattern in IL-1beta and TNFalpha promoters of myeloid precursors, monocytic cells and PMN was comparable. Activation of IL-1beta and TNFalpha expression in zinc deficient promyeloid cells also seems to be caused by the opening of their proximal promoters. Interestingly, intracellular zinc concentrations are decreased during monopoiesis. Since HDAC-activity is zinc-dependent a connection between intracellular zinc concentrations, HDAC activity and IL-1beta as well as TNFalpha promoter structures is likely. Moreover, results of this study proved that the production of reactive oxygen species by promyeloid cells is increased during zinc deficiency, supporting the p38-dependent post-transcriptional processing of IL-1beta and TNFalpha. In conclusion results from this thesis show that histone acetylation and chromatin remodeling form the basis for physiological activation of IL-1beta and TNFalpha expression during monopoiesis as well as for deregulation during zinc deficiency. Those data might enable the development of new epigenetic and zinc-based therapeutical approaches to treat IL-1beta or TNFalpha deregulation

Epigenetische Veränderungen in den IL-1beta und TNFalpha Promotoren während der Zelldifferenzierung und unter Zinkmangel

IL-1beta and TNFalpha are involved in a variety of immunological processes. Their deregulation can cause immunosuppression or chronic inflammation. Both cytokines are primarily synthesized by monocytes but not neutrophil granulocytes (PMN) or their common haematopoietic precursors (HPC), as demonstrated in this thesis. Via in vitro differentiation of HPC it could be shown for the first time in primary cells that the proximal IL-1beta promoter is remodeled into an open structure during monopoiesis but not granulopoiesis. In contrast, chromatin remodeling of the TNFalpha promoter was stimulation dependent, but restricted to monocytes, only. No changes in TNFalpha promoter structure were detected in stimulated PMN or HPC. Basis for the chromatin remodeling of IL-1beta and TNFalpha promoters is presumably their hyperacetylation, simulated by inhibition of histone deacetylases (HDAC) herein. Specific DNA- or histone methylation of IL-1beta and TNFalpha promoter during differential activation of IL-1beta and TNFalpha expression of monocytes and PMN could be excluded. However, an indirect effect due to methylation dependent activation of IL-1beta and TNFalpha regulating genes is assumable. The involvement of the myeloid transcription factors PU.1 and C/EBPbeta is also unlikely, since their binding pattern in IL-1beta and TNFalpha promoters of myeloid precursors, monocytic cells and PMN was comparable. Activation of IL-1beta and TNFalpha expression in zinc deficient promyeloid cells also seems to be caused by the opening of their proximal promoters. Interestingly, intracellular zinc concentrations are decreased during monopoiesis. Since HDAC-activity is zinc-dependent a connection between intracellular zinc concentrations, HDAC activity and IL-1beta as well as TNFalpha promoter structures is likely. Moreover, results of this study proved that the production of reactive oxygen species by promyeloid cells is increased during zinc deficiency, supporting the p38-dependent post-transcriptional processing of IL-1beta and TNFalpha. In conclusion results from this thesis show that histone acetylation and chromatin remodeling form the basis for physiological activation of IL-1beta and TNFalpha expression during monopoiesis as well as for deregulation during zinc deficiency. Those data might enable the development of new epigenetic and zinc-based therapeutical approaches to treat IL-1beta or TNFalpha deregulation

Zinc Signals and Immunity

Zinc homeostasis is crucial for an adequate function of the immune system. Zinc deficiency as well as zinc excess result in severe disturbances in immune cell numbers and activities, which can result in increased susceptibility to infections and development of especially inflammatory diseases. This review focuses on the role of zinc in regulating intracellular signaling pathways in innate as well as adaptive immune cells. Main underlying molecular mechanisms and targets affected by altered zinc homeostasis, including kinases, caspases, phosphatases, and phosphodiesterases, will be highlighted in this article. In addition, the interplay of zinc homeostasis and the redox metabolism in affecting intracellular signaling will be emphasized. Key signaling pathways will be described in detail for the different cell types of the immune system. In this, effects of fast zinc flux, taking place within a few seconds to minutes will be distinguish from slower types of zinc signals, also designated as “zinc waves”, and late homeostatic zinc signals regarding prolonged changes in intracellular zinc

Parameters Influencing Zinc in Experimental Systems in Vivo and in Vitro

In recent years, the role of zinc in biological systems has been a subject of intense research. Despite wide increase in our knowledge and understanding of zinc homeostasis, numerous questions remain to be answered, encouraging further research. In particular, the quantification of intracellular zinc ions and fluctuation, as well as the function of zinc in signaling processes are being intensely investigated. The determination of free intracellular zinc ions is difficult and error-prone, as concentrations are extremely low (in the pico- to nanomolar range), but techniques exist involving fluorescent probes and sensors. In spite of zinc deficiency being accepted as a global problem, causing death and disease worldwide, to date there are no markers to reliably assess a person’s zinc status. This review summarizes the difficulties and major pitfalls when working with zinc in in vitro and in vivo research. Additionally, it specifies important aspects for zinc substitution and supplementation, including the bioavailability of zinc and its intestinal absorption. In particular, it is intended to help researchers with yet minor experience working with zinc efficiently set up experiments and avoid commonly occurring mistakes, starting with the choice and preparation of reagents and instrumentation, and concluding with possibilities for measuring the status of zinc in humans